PCR: The Polymerase Chain Reaction
PCR is a technique used to amplify the number of copies of a specific region of DNA in order to produce enough DNA to be adequately tested. PCR has become one of the most widely used techniques in molecular biology.
It is a rapid and simple means of producing relatively large numbers of copies of DNA molecules from minute quantities of source DNA material.
THE PRINCIPLE OF PCR
The purpose of PCR is to make a huge number of copies of a gene. This is necessary to have enough starting template for sequencing.
THE BASIC STEPS OF PCR
There are three basic steps in a PCR, which are repeated for 30 or 40 cycles. This is done on an automated cycler, which can heat and cool the tubes with the reaction mixture in a
very short time.
The basic PCR steps are:
- DENATURATION (at 94 degrees C). During this step, the double strand melts open to single stranded DNA, and all enzymatic reactions stop.
- HYBRIDIZATION or ANNEALING (at 54 degrees C). At this step, the single-stranded sample DNA is “mixed” with a probe, and under the right conditions, hydrogen bonds form between this probe and its complementary
sequence in the sample DNA. The double-helix structure is re-created.
- DNA SYNTHESIS or REPLICATION (at 72 degrees C). This step presents the ideal temperature for the polymerase. Here, the primer (a piece of DNA that is base paired to the template strand in such a way that the 3’
end of it is available to serve as the starting point for the new DNA) starts the process of “multiplying” the amount of DNA by attracting bases to attach to it. Primers that are on positions with no exact match
get loose again and don’t give an extension of the fragment.
PCR is a clever procedure that takes advantage of DNA polymerase enzymes and synthetic oligonucleotides to make many copies of a specific fragment of
DNA. And because the reaction mixture contains primers complementary to both strands of DNA, the products of the DNA synthesis can themselves be copied with the opposite primer.